The end-labeling of ribonucleic acids is fundamental to a variety of molecular biology applications. Applications in which RNA is end-labeled include: hybridization assays, in which the probe employed is an end-labeled ribonucleic acid of the subject invention, e.g. Southern analyses, northern analyses, DNA library screens, in situ hybridization experiments, e.g. chromosome squashes, tissue sections, ctc.; sequencing applications, e.g. RNA direct chemical sequencing methods; hybridizations of labeled RNAs to chips/arrays; and the like.
A number of different protocols have been developed for 3'-end labeling RNA. In a first method that has been reported in the literature, bacteriophage T4 RNA ligase is used to attach radioactively labeled ribonucleotides to the 3' end of RNA molecules. Such ligase reactions typically require long incubation times to achieve sufficient labeling, e.g. 12 hours. In another method, terminal deoxynucleotidyl transferase (TdT) is employed to attach labeled deoxyribonucleotides to the 3' ends of RNAs. Disadvantages of this method include the fact that TdT cannot label with ribonucleotides (i.e. it cannot use ribonucleotides as donors) and that TdT uses both DNA and RNA as a substrate. Finally, poly(A) polymerase has been employed to label RNAs with radioactively labeled ribonucleotides. Disadvantages with this protocol include the use of radioactively labeled materials.
As such, despite the number of different protocols that have been developed for the 3' end-labeling of ribonucleic acids, there continues to be interest in the development of new methods for end-labeling ribonucleic acids. Of particular interest would be the development of a method of end-labeling ribonucleic acids which was able to rapidly and efficiently label a ribonucleic acid with non-radioactively labeled ribonucleotides, particularly fluorescently labeled ribonucleotides.